The present invention relates generally to methods of analysis and investigation which utilize a solid support in the form of membranes or similar media upon which selected specimens are transferred to or otherwise placed for analysis and evaluation. More particularly, the present invention relates to and is suited for use in those areas of biotechnology and molecular biology that utilize membranes upon which selected specimens are deposited for analysis, investigation, hybridization, and the like, such specimens including molecules and molecule fragments of DNA, RNA, and proteins.
Many laboratory and analytical procedures involve the use of a sheet-like membrane, such as nitrocellulose, treated nitrocellulose, and similar materials, upon which one or more specimens are deposited with the membrane then subjected to further processing steps to analyze, identify, or isolate selected of the specimens. For example, in the investigation of nucleic acids, the study of the structure and characteristics of DNA and RNA, and the function of selected enzymes in dividing DNA and RNA molecules into fragments of varying size, the use of sheet-like membranes, particularly those of nitrocellulose, are central to isolating selected fragments having certain characteristics. Various membrane-utilizing processes have been developed for the investigation of nucleic acids; these processes have in common the step of transferring or otherwise depositing DNA or RNA specimens onto a membrane. The membrane is then subjected to subsequent processing in accordance with the particular methodology of the process. For example, in one process, termed the "Southern" blot procedure, fragments of DNA molecules of unlike size are electrophoretically separated into groupings of similar size. The fragments are then transferred to a nitrocellulose membrane for subsequent processing to produce a visible indication, for example, by autoradiograph, of the position on the membrane of the target fragments. In another process, termed the "dot" blot procedure, fragments of DNA molecules of unlike size are separated, for example, by ultra-centrifuging or column chromatography, into separate samples of like size. The separate samples are then deposited onto a nitrocellulose membrane with each sample occupying a dot-like area on the membrane. The membrane is then subjected to additional processing steps to again yield an autoradiograph which indicates the position of the target fragments on the membrane.
The present invention can be best understood in the context of the Southern blot procedure, selected steps of which are illustrated in FIGS. 1, 2, and 3. In the Southern blot procedure, as well as the other membrane utilizing procedures, source DNA molecules are cleaved into fragments of differing length by restriction enzymes that recognize selected sites on the DNA molecule and cleave the molecule at those recognition sites into molecule fragments of varying lengths. As part of the analytical process, the molecular fragments are separated as a function of their length. In the Southern blot procedure, the molecular fragments are electrophoretically separated. As schematically illustrated in FIG. 1, an agarose or polyacrylamide gel slab G is prepared with electrodes, schematically illustrated at E1 and E2, aligned along opposite edges of the slab. The source mixture of molecule fragments is deposited on the slab G and the electrodes are connected to a suitable source of electrical energy to apply a directed electric field across the gel slab between the two electrodes. Since the molecule fragments have a net charge, they will migrate through and across the slab G toward the oppositely charged electrode with the speed of transportation being a function, in part, of the molecular weight of the fragment. In time, molecules of similar size will be grouped with one another in spaced apart, band-like groupings with the largest fragments grouped relatively close to the initial position and the smallest fragments grouped furthest from the initial position to thus effectively separate the molecules as a function of size.
After the molecule fragments have been electrophoretically separated as a function of fragment size, the fragments are transferred from the gel to a membrane M having an affinity for the particular molecule fragments. Where DNA fragments have been separated in the gel slab G, a sheet-like membrane M of nitrocellulose is laid upon one surface of the gel. Where other molecules, such as RNA are separated, another membrane having an affinity to RNA, such as diazobenzyloxymethyl cellulose (DBM) paper or aminophenylthioether (APT) paper activated to the diazo-form (DPT), may be used.
Once the membrane M has been applied to the gel slab G, the molecules in the gel matrix can be transferred to the membrane M by the Southern transfer method by establishing a capillary transfer through the gel and the contiguous membrane M. The gel slab G and the membrane M are placed upon the upper surface of an absorbent material S, which may take the form of a stack of blotting papers, saturated with a blotting buffer solution. A dry absorbent material D, which may also take the form of a stack of absorbent blotting sheets, is placed on the upper side of the membrane M so that a capillary transfer is established from the saturated material S through the gel slab G and the membrane M to the dry or unsaturated absorbent material. As the blottng buffer passes from the saturated to the unsaturated materials, the electrophoretically separated DNA molecules are eluted from the gel matrix and transferred to the membrane M with the molecular fragments binding to the membrane. This transfer process can be assisted electrophoretically by establishing an electric field across the absorber stack to assist in moving the molecules from the gel matrix to the membrane M. Regardless of the particular gel-to-membrane transfer mechanism employed, the resulting membrane M will have groupings of DNA molecule fragments bound thereto.
The membrane M is then subjected to a number of fluid treatment steps to identify a particular grouping of target DNA fragments on the membrane. Typically, the transferred DNA fragments are thermally "fixed" to the membrane M by heating at a selected temperature for a period of time sufficient to effect fixing. In order to locate a group of particular target fragments bound to the membrane M, a solution of DNA or RNA "probe" fragments complementary to the target fragments is prepared with the probe fragments coupled to a radioactive tracer material. The membrane M is then washed in the probe solution, for example, by immersion in a capped bottle or heat-sealed plastic bag containing the probe solution, for an incubation period sufficient to allow the radio-tagged probe fragments to hybridized with their complementary target fragments on the membrane. Once sufficient time for annealing has lapsed, the membrane M is then washed and treated in a series of buffer solutions, such as ribonuclease, at differing temperatures and concentrations designed to remove the excess unhybridized probe solution. The resulting membrane M is dried and retains only the original DNA fragments and the hybridized probe and target radio-tagged fragments. Thereafter, the membrane M is processed to yield a visible indication of the location of the annealed target/probe molecules. Typically, the visible indication is obtained by laying the membrane against one side of a radiation sensitive film so that the film is exposed by beta particle radiation from the radioactive tag. The location of the hybridized probe/target molecule fragments on the membrane M is revealed by the developed film.
Throughout the above described processing steps, the typically thin (e.g. 0.001 inch) and structurally weak membrane is subject to many manual handling steps over a relatively long period of time. Even where higher strength membranes are available, conventional membrane-dependent procedures require a rather high level of skill to insure valid and reproducible results and to minimize physical damage to or contamination of the membrane. Also, the use of membranes is not conducive to time and cost efficiencies that would allow transfer of the membrane-based methodologies to clinical, industrial, and agricultural applications, or other applications where cost and time effectiveness is imperative.